Asef is a protein that was found by the present inventors as a colorectal tumor suppressor gene-associated protein M1, which has already been disclosed and for which a patent application has been filed (Patent Reference 1 and Non-patent Reference 1). The protein consists of 619 amino acid residues, and contains the Db1 homology (DH) domain, the pleckstrin homology (PH) domain and the Src homology 3 (SH3) domain in its amino acid sequence.
In terms of function, it is known that Asef has GEF activity specific for Rac. Rac belongs to the Rho family, which is one of the small GTP-binding protein families. More specifically, Asef binds to Rac to stimulate a GDP/GTP exchange reaction which results in the activation of Rac, thereby acting on NFκB, c-jun, SRE and the like, which are located downstream of the Rac related-intracellular signal transduction. The Rho family proteins play key roles in the reorganization of the acting network, thereby regulating cell migration and cell-cell adhesion. Therefore, there is a possibility that Asef induces cellular lamellipodia (lobopodium) or cell membrane ruffling and participates in cell migration and cell-cell adhesion.
It has been revealed that the binding of Asef to the gene product of the tumor suppressor gene APC via the armadillo repeat domain of the gene product. The GEF activity of Asef is positively regulated by the APC gene product. Actually, the induction of Asef-mediated cell membrane ruffling or lamellipodia formation by the APC gene product is observed in MDCK cells that are canine kidney-derived epithelial-like cells. Further, Asef accumulates at the tips of microtubules in motile cells similarly to the APC gene product. Therefore, Asef may hold the key to control cell migration when cells migrate from the crypt to the villus tip of the colon.
Meanwhile, the tumor suppressor gene APC (Non-patent Reference 2) has been isolated as a responsible gene for familial adenomatous polyposis (FAP). Mutation of the gene is observed in approximately 70% to 80% of sporadic colorectal cancers. The APC gene product (hereunder, referred to as “APC”) is a giant protein of approximately 300 kDa that comprises 2,843 amino acid residues. APC contains an armadillo repeat domain in the amino acid sequence thereof that participates in protein-protein interaction. Most somatic APC mutations observed in colorectal tumor cells occur within its central region called the “mutation cluster region (MCR)” and result in the generation of truncated APCs that lack the binding sites for microtubules, EB 1 or hDLG, and at least some of the sites for β-catenin and Axin (Non-patent References 4, 5, 6, 7 and 8). However, the region of APC responsible for binding to Asef, the armadillo repeat domain, is retained in most mutant APCs (Non-patent References 6, 7 and 8). APC has a function to bind to β-catenin, one kind of oncogene product, to induce its degradation (Non-patent References 2, 3, 4, 5 and 6). β-catenin, which is a Wnt/Wingless signal transduction factor, binds to the cytoplasmic domain of cadherin and plays a role in cell adhesion, while it plays important roles in developmental processes and in tumorigenesis (Non-patent References 9 and 10).
The amino acid sequence of Asef and the nucleotide sequence of its gene have been deposited with GenBank under the accession number AB042199. Further, the amino acid sequence of APC and the nucleotide sequence of its gene have been deposited with GenBank under the accession number NM000038.
Documents referred to in this specification are listed hereunder:    Patent Reference 1: Japanese Patent Laid-Open No. 2001-057888.    Non-patent Reference 1: Kawasaki, Y., et al., Science, 2000, Vol. 289, p. 1194-1197.    Non-patent Reference 2: Kinzler, K. W., et al., Cell, 1996, Vol. 87, p. 159-170.    Non-patent Reference 3: Fearnhead, et al., Human Molecular Genetics, 2001, Vol. 10, p. 721-733.    Non-patent Reference 4: Bienz, M., et al., Cell, 2000, Vol. 103, p. 311-320.    Non-patent Reference 5: Perifer, M., et al., Science, 2000, Vol. 287, p. 1606-1609.    Non-patent Reference 6: Akiyama, T., Cytokine and Growth Factor Reviews, 2000, Vol. 11, p. 273-282.    Non-patent Reference 7: Miyoshi, Y., et al., Human Molecular Genetics, 1992, Vol. 1, p. 229-233.    Non-patent Reference 8: Nagawa, H., et al., Human Mutation, 1993, Vol. 2, p. 425-434.    Non-patent Reference 9: Cell, 1996, Vol. 86, p. 391-399.    Non-patent Reference 10: Nature, 1996, Vol. 382, p. 638-642.    Non-patent Reference 11: Wong, M. H., et al., Proceeding of national academy of science USA┘ 1996, Vol. 93, p. 9588-9593.    Non-patent Reference 12: Oshima, H., et al., Cancer Research, 1997, Vol. 57, p. 1644-1649.    Non-patent Reference 13: Paddison, P. J., et al., Genes and Development, 2002, Vol. 16, p. 948-958.